Native tertiary structure in an A-state.
The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with native secondary structure and non-native, fluctuating tertiary structure. We used yeast iso-1-ferricytochrome c to test for an evolutionary-invariant tertiary interaction in its A-state. Thermal denaturation monitored by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities, delta GA reversible D and delta GN reversible D. We examined the wild-type protein, seven variants with substitutions at the interface between the N and C-terminal helices, and four control variants. The controls have the same amino acid changes as the interface variants, but the changes are close to, not at, the interface. We also examined the pH and sulfate concentration dependencies and found that while these factors affect the far-UV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants. A delta GA reversible D versus-delta GN reversible D plot for the interface variants has a slope near unity and the control variants have near-wild-type stability. These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, confirming our preliminary report. We determined that the heat capacity change for A-state denaturation is approximately 60% of the value for native-state denaturation, indicating that the A-state interior is native-like. We discuss our results in relation to ferricytochrome c folding kinetics.
Marmorino
J L
JL
Department of Chemistry, University of North Carolina at Chapel Hill 27599-3290, USA.
Lehti
M
M
Pielak
G J
GJ
D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D019143:Evolution, Molecular; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011048:Polarography; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c